A comprehensive qualitative investigation of the factors that affect surgical site infection prevention in cardiac surgery in England using observations and interviews.

BACKGROUND: Interview and questionnaire studies have identified barriers and challenges to preventing surgical site infections (SSIs) by focusing on compliance with recommendations and care bundles using interviews, questionnaires and expert panels. This study proposes a more comprehensive investigation by using observations of clinical practice plus interviews which will enable a wider focus. AIM: To comprehensively identify the factors which affect SSI prevention using cardiac surgery as an exemplar. METHODS: The study consisted of 130 h of observed clinical practice followed by individual semi-structured interviews with 16 surgeons, anaesthetists, theatre staff, and nurses at four cardiac centres in England. Data were analysed thematically. FINDINGS: The factors were complex and existed at the level of the intervention, the individual, the team, the organization, and even the wider society. Factors included: the attributes of the intervention; the relationship between evidence, personal beliefs, and perceived risk; power and hierarchy; leadership and culture; resources; infrastructure; supplies; organization and planning; patient engagement and power; hospital administration; workforce shortages; COVID-19 pandemic; 'Brexit'; and the war in Ukraine. CONCLUSION: This is one of the first studies to provide a comprehensive overview of the factors affecting SSI prevention. The factors are complex and need to be fully understood when trying to reduce SSIs. A strong evidence base was insufficient to ensure implementation of an intervention.

Barriers and facilitators for surgical site infection surveillance for adult cardiac surgery in a high-income setting: an in-depth exploration.

BACKGROUND: Surgical site infection (SSI) surveillance aims to facilitate a reduction in SSIs through identifying infection rates, benchmarking, triggering clinical review and instituting infection control measures. Participation in surveillance is, however, variable suggesting opportunities to improve wider adoption. AIM: To gain an in-depth understanding of the barriers and facilitators for SSI surveillance in a high-income European setting. METHODS: Key informant interviews with 16 surveillance staff, infection prevention staff, nurses and surgeons from nine cardiac hospitals in England. Data were analysed thematically. FINDINGS: SSI surveillance was reported to be resource intensive. Barriers to surveillance included challenges associated with data collection: data being located in numerous places, multiple SSI data reporting schemes, difficulty in finding denominator data, lack of interface between computerized systems, 'labour intensive' or 'antiquated' methods to collect data (e.g., using postal systems for patient questionnaires). Additional reported concerns included: relevance of definitions, perceived variability in data reporting, lack of surgeon engagement, unsupportive managers, low priority of SSIs among staff, and a 'blame culture' around high SSI rates. Facilitators were increased resources, better use of digital technologies (e.g., remote digital wound monitoring), integrating surveillance within routine clinical work, having champions, mandating surveillance, ensuring a closer relationship between surveillance and improved patient outcomes, increasing the focus on post-discharge surveillance, and integration with primary care data. CONCLUSION: Using novel interviews with 'front-line' staff, identified opportunities for improving participation in SSI surveillance. Translating these findings into action will increase surveillance activity and bring patient safety benefits to a larger pool of surgical patients.

A High-throughput Screening of a Chemical Compound Library in Ovarian Cancer Stem Cells.

BACKGROUND: Epithelial ovarian cancer has a poor prognosis, mostly due to its late diagnosis and the development of drug resistance after a first platinum-based regimen. The presence of a specific population of "cancer stem cells" could be responsible of the relapse of the tumor and the development of resistance to therapy. For this reason, it would be important to specifically target this subpopulation of tumor cells in order to increase the response to therapy. METHOD: We screened a chemical compound library assembled during the COST CM1106 action to search for compound classes active in targeting ovarian stem cells. We here report the results of the high-throughput screening assay in two ovarian cancer stem cells and the differentiated cells derived from them. RESULTS AND CONCLUSION: Interestingly, there were compounds active only on stem cells, only on differentiated cells, and compounds active on both cell populations. Even if these data need to be validated in ad hoc dose response cytotoxic experiments, the ongoing analysis of the compound structures will open up to mechanistic drug studies to select compounds able to improve the prognosis of ovarian cancer patients.

Bypassing the proline/thiazoline requirement of the macrocyclase PatG.

Biocatalysis is a fast developing field in which an enzyme's natural capabilities are harnessed or engineered for synthetic chemistry. The enzyme PatG is an extremely promiscuous macrocyclase enzyme tolerating both non-natural amino acids and non-amino acids within the substrate. It does, however, require a proline or thiazoline at the C-terminal position of the core peptide which means the final product must contain this group. Here, we show guided by structural insight we have identified two synthetic routes, triazole and a double cysteine, that circumvent this requirement. With the triazole, we show PatGmac can macrocyclise substrates that do not contain any amino acids in the final product.

Synthetic studies on the bioactive tetramic acid JBIR-22 using a late stage Diels-Alder reaction.

A late stage Diels-Alder reaction is used to prepare a mixture of JBIR-22, a natural product from the Equisetin family of tetramic acids, and one of its diastereomers. This is achieved in just 8 steps from pyruvate. The success of the late stage DA approach is discussed in the context of the biosynthesis of JBIR-22 (and perhaps related natural products).

p53 and cell cycle independent dysregulation of autophagy in chronic lymphocytic leukaemia.

BACKGROUND: Activation of wild-type p53 with the small molecule sirtuin inhibitor Tenovin-6 (Tnv-6) induces p53-dependent apoptosis in many malignant cells. In contrast, Tnv-6 reduces chronic lymphocytic leukaemia (CLL) cell viability with dysregulation of autophagy, without increasing p53-pathway activity. METHODS: Here, we have investigated whether a quiescent phenotype (unique to CLL) determines the Tnv-6 response, by comparing the effects of Tnv-6 on activated and proliferating CLL. We further studied if these responses are p53-dependent. RESULTS: Unlike quiescent cells, cell death in activated cultures treated with Tnv-6 was consistently associated with p53 upregulation. However, p53 acetylation remained unchanged, without caspase-3 cleavage or apoptosis on electron microscopy. Instead, cellular ultrastructure and protein profiles indicated autophagy inhibition, with reduced ubiquitin-proteasome activity. In specimens with mutant TP53 cultured with Tnv-6, changes in the autophagy-associated protein LC3 occurred independently of p53. Cells treated with Tnv-6 analogues lacking sirtuin inhibitory activity had attenuated LC3 lipidation compared with Tnv-6 (P0.01), suggesting that autophagy dysregulation occurs predominantly through an effect on sirtuins. CONCLUSION: These cell cycle and p53-independent anti-leukaemic mechanisms potentially offer novel therapeutic approaches to target leukaemia-sustaining cells in CLL, including in disease with p53-pathway dysfunction. Whether targets in addition to sirtuins contribute to autophagy dysregulation by Tnv-6, requires further investigation.

Review article: what do patients with inflammatory bowel disease want for their clinical management?

BACKGROUND: The patient's perceptions of benefits and risks of treatment differ from those of physicians, yet this topic is rarely explored in ulcerative colitis or Crohn's disease. AIM: To present a patient's perspective on care for their colitis, to consider how patients' views will influence preliminary proposals for standards of care, and to discuss the different priorities of patients and physicians. METHODS: Description of an individual patient's journey from diagnosis of ulcerative colitis to colectomy, compared with priorities proposed by a patient-based organization (National Association of Colitis and Crohn's disease). RESULTS: Awareness of colitis in primary care, prompt referral, timely investigation and specialist care if admitted to hospital are shared goals of patients and physicians. Patients are generally prepared to accept higher risks from medical treatment to avoid their undesirable outcomes (especially colectomy) than physicians will on their behalf. There has been little systematic study of this disparity in ulcerative colitis. Under-reporting the symptom burden, tolerance of treatment side effects, substantial lifestyle changes and willingness to explore any treatment options are characteristic coping strategies. Physicians should be alert to coping strategies, with a willingness to seek second opinions. CONCLUSION: Perceptions of risk and benefit differ between patients and physicians, and recognition of this difference is a starting point for difficult clinical decision-making.

Targeting invasion and egress: from tools to drugs?

A recent resurgence in the use of compounds to study essential biological processes raises important questions concerning the link between fundamental research and drug development. This article discusses many of the issues involved, in the context of host cell invasion and egress by parasites of the Phylum Apicomplexa. In addition, an overview of the key steps in invasion and egress is provided with a particular emphasis on potential parasite protein drug targets.

Differentially expressed genes in adult familial myelodysplastic syndromes.

The precise genetic events leading to myelodysplastic syndromes (MDSs) and leukemic transformation remain poorly defined. Even less is known about adult familial MDS. We report an adult MDS family in whom enriched tissue-specific transcripts were derived by subtractive hybridization of cDNA from the mononuclear and CD34+ cells of affected and unaffected family members. These expression libraries were then hybridized to Genome Discovery arrays containing 18 404 genes and expressed sequence tags, and several clusters of differentially expressed genes were identified. A group of 21 genes was underexpressed (>5-fold) in affected vs unaffected family members, and among these were transcription factors and genes involved in myeloid differentiation, such as ZNF140 and myeloid nuclear differentiation antigen (MNDA). Another group of 36 genes was overexpressed (>5-fold), and these encoded proteins belonging to signaling pathways, such as Ras- and Fos-related genes. The top two genes downregulated in this MDS family, ZNF140 and MNDA, were similarly altered in another MDS family, and in some cases of sporadic MDS. Our data suggest that we have identified genes differentially expressed in adult familial MDS, and that alteration of some of these genes may also be important for the evolution of different stages or severity of sporadic MDS.

Screening and synthesis: high throughput technologies applied to parasitology.

High throughput technologies continue to develop in response to the challenges set by the genome projects. This article discusses how the techniques of both high throughput screening (HTS) and synthesis can influence research in parasitology. Examples of the use of targeted and phenotype-based HTS using unbiased compound collections are provided. The important issue of identifying the protein target(s) of bioactive compounds is discussed from the synthetic chemist's perspective. This article concludes by reviewing recent examples of successful target identification studies in parasitology.

Polycythemia vera: analysis of DNA from blood granulocytes using comparative genomic hybridization.

BACKGROUND AND OBJECTIVES: The diagnosis of polycythemia vera (PV) is supported by the finding of an abnormal karyotype in patients with erythrocytosis. However, most PV patients have normal marrow cytogenetics at presentation and there is reluctance to use this test routinely. Comparative genomic hybridization (CGH) is a cytogenetic screening technique that analyzes interphase cells. This approach offers practical advantages over conventional cytogenetics and interphase fluorescence in-situ hybridization (IFISH). We have therefore evaluated the diagnostic utility of CGH applied to blood granulocytes in PV. DESIGN AND METHODS: Blood granulocytes from 17 PV patients were analyzed using CGH and the results compared with those from previous conventional cytogenetics and IFISH studies. RESULTS: Three patients had abnormal CGH profiles. One case had gain of 9p. This patient had normal IFISH results using a centromere-9 probe. The second case had complete gain of chromosomes 8 and 9 and the third had complete gain of chromosome 9, all confirmed by IFISH: Cytogenetics had not been performed in two of these cases and had failed in the third. Three cases with 20q deletion according to cytogenetics and/or IFISH, were normal by CGH. The remaining subjects were normal by all methods. INTERPRETATION AND CONCLUSIONS: CGH analysis of blood granulocytes can detect the chromosome gains commonly observed in PV. However, CGH cannot be relied on to detect 20q deletions, which are the most frequent cytogenetic abnormality in PV. Thus, CGH has a role in the diagnosis and follow-up of PV patients, but must be used in conjunction with other methods.

An alkylsilyl-tethered, high-capacity solid support amenable to diversity-oriented synthesis for one-bead, one-stock solution chemical genetics.

The synthesis and use of an alkylsilyl-tethered large (500-600 microm) polystyrene resin (1) are disclosed. An optimized Suzuki coupling of bromine-functionalized polystyrene and a silicon-functionalized alkylborane generates the silicon-substituted polystyrene 1 in large scale (>100 g). Resin loading is accomplished by activation as the silyl triflate, which can accommodate even sterically encumbered secondary alcohols and phenols. Treatment with HF/pyridine for linker cleavage is mild, efficient, and amenable to an automated, large-scale distribution system. This platform delivers, minimally, 50 nmol of each small molecule derived from a diversity-oriented, split-pool synthesis on a per bead basis for use in both forward and reverse chemical genetic assays. This technology satisfies many requirements of a one bead-one stock solution approach to chemical genetics.

The incidences of trisomy 8, trisomy 9 and D20S108 deletion in polycythaemia vera: an analysis of blood granulocytes using interphase fluorescence in situ hybridization.

We have used interphase fluorescence in situ hybridization (IFISH) to detect trisomy 8, trisomy 9 and 20q deletion in circulating granulocytes from patients with polycythaemia vera (PV). Out of 64 PV patients, 15 (23%) exhibited an abnormality. Two patients had trisomy 9, three had trisomy 8 and 10 patients had hemizygous deletion of D20S108 (a locus in the 20q common deleted region). Aberrant nuclei ranged from 10% to 80% in these 15 cases. There was no correlation between the presence of a marker and sex, age, interval between presentation and IFISH analysis, neutrophil or platelet count or therapy. Conventional marrow cytogenetic karyotype results were available in 23 cases and there was concurrence between these and blood IFISH in 16 cases (13 normal and three with 20q/D20S108 deletion by both methods). Three patients with D20S108 deletion by IFISH were normal by previous marrow cytogenetic testing and four cases with 20q deletion by previous marrow cytogenetics had normal blood granulocytes according to IFISH. Thus, we confirm that trisomies 8 and 9 and deletion of 20q are diagnostically useful markers of PV. IFISH analysis of blood granulocytes is a practical method for detecting these markers, but as an adjunct to, not as a substitute for, conventional marrow cytogenetics.

Mechanistic insights into the inhibition of serine proteases by monocyclic lactams.

Although originally discovered as inhibitors of pencillin-binding proteins, beta-lactams have more recently found utility as serine protease inhibitors. Indeed through their ability to react irreversibly with nucleophilic serine residues they have proved extraordinarily successful as enzyme inhibitors. Consequently there has been much speculation as to the reason for the general effectiveness of beta-lactams as antibacterials or inhibitors of hydrolytic enzymes. The interaction of analogous beta- and gamma-lactams with a serine protease was investigated. Three series of gamma-lactams based upon monocyclic beta-lactam inhibitors of elastase [Firestone, R. A. et al. (1990) Tetrahedron 46, 2255-2262.] but with an extra methylene group inserted between three of the bonds in the ring were synthesized. Their interaction with porcine pancreatic elastase and their efficacy as inhibitors were evaluated through the use of kinetic, NMR, mass spectrometric, and X-ray crystallographic analyses. The first series, with the methylene group inserted between C-3 and C-4 of the beta-lactam template, were readily hydrolyzed but were inactive or very weakly active as inhibitors. The second series, with the methylene group between C-4 and the nitrogen of the beta-lactam template, were inhibitory and reacted reversibly with PPE to form acyl-enzyme complexes, which were stable with respect to hydrolysis. The third series, with the methylene group inserted between C-2 and C-3, were not hydrolyzed and were not inhibitors consistent with lack of binding to PPE. Comparison of the crystal structure of the acyl-enzyme complex formed between PPE and a second series gamma-lactam and that formed between PPE and a peptide [Wilmouth, R. C., et al. (1997) Nat. Struct. Biol. 4, 456-462.] reveals why the complexes formed with this series were resistant to hydrolysis and suggests ways in which stable acyl-enzyme complexes might be obtained from monocyclic gamma-lactam-based inhibitors.

Inhibition of elastase by N-sulfonylaryl beta-lactams: anatomy of a stable acyl-enzyme complex.

beta-Lactam inhibitors of transpeptidase enzymes involved in cell wall biosynthesis remain among the most important therapeutic agents in clinical use. beta-Lactams have more recently been developed as inhibitors of serine proteases including elastase. All therapeutically useful beta-lactam inhibitors operate via mechanisms resulting in the formation of hydrolytically stable acyl-enzyme complexes. Presently, it is difficult to predict which beta-lactams will form stable acyl-enzyme complexes with serine enzymes. Further, the factors that result in the seemingly special nature of beta-lactams versus other acylating agents are unclear-if indeed they exist. Here we present the 1.6 A resolution crystal structure of a stable acyl-enzyme complex formed between porcine pancreatic elastase and a representative monocyclic beta-lactam, which forms a simple acyl-enzyme. The structure shows that the ester carbonyl is not located within the oxyanion hole and the "hydrolytic" water is displaced. Combined with additional kinetic and mass spectrometric data, the structure allows the rationalization of the low degree of hydrolytic lability observed for the beta-lactam-derived acyl-enzyme complex.